Castilho Leda R., Moraes Angela Maria (Ed.) Animal Cell Technology: From Biopharmaceuticals to Gene Therapy

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474 Animal Cell Technology

Cell therapies and stem

cells

Hamilton da Silva Jr and Radovan Borojevic

20.1 Introduction

Cell therapy is a medical procedure that rebuilds structures and restores

functions of deteriorated tissues or organs by stem cells. This approach

is feasible in treatments of tissue dysfunction generated by trauma,

infectious disease, degenerative processes caused by natural fatigue or

environmental insults, or a natural consequence of senescence. The cell-

mediated regenerative medical interventions have a number of technical

and biological limitations, which have stimulated new studies in bio-

logical processes and engineering. The advances in these two domains

have created a multidisciplinary new ﬁeld of biotechnology and bioengi-

neering. The innovative work in this ﬁeld has the potential for improv-

ing the quality of life of humans and signiﬁcantly decreasing healthcare

costs. The traditional use of cell therapy has been in bone marrow

transplantation and treatment of autoimmune diseases. More recently,

cell therapies have been applied with success in reparative surgery of

soft tissues, skin and mucosa, peripheral nerve lesions, orthopedics

including degenerative processes and trauma, angiogenesis, heart dis-

eases, and stroke.

The application of cell therapies is based on histological (homotopic

or heterotopic) and immunological (autologous transplants, allotrans-

plants or xenotransplants) principles. Homotopic therapies are deﬁned

as procedures that use cells for repair and regeneration of the tissue

from which they have been harvested. Good examples of homotopic

therapies are: skin repair with in vitro expanded epidermal cells, isola-

tion and implantation of pancreatic Langerhans islets (Eliaschewitz et

al., 2004), and bone marrow aspiration to restore the hematopoietic

tissue (Shizuru et al., 2005). Heterotopic therapies use stem cells

obtained from a source different from that in which they are used. The

most frequent source of stem cells used in repair of peripheral tissues is

bone marrow. However, it is now known that many tissues, such as

adipose or blood vessel walls, harbor pluripotent stem cells that can be

used in repair and regeneration. This procedure depends upon the

pluripotency of stem cells or their transdifferentiation, and the under-

lying molecular mechanisms are not well understood. The paracrine

effect of implanted cells that may activate the resident cell progenitors

has to be taken into consideration, as well as modiﬁcation of the

intercellular environment following cell injection.

Autologous transplants involve the transfer of cells, tissues, or organs

from the same individual. Conversely, allotransplants or allogeneic

transplants use cells from different individuals within the same species.

Xenotransplants involve transplantation or temporary use of organs or

tissues from a different species (e.g. porcine to human). In cases of

allotransplants and xenotransplants, the implanted cells are necessarily

identiﬁed as foreign by the receptor’s immune system, which triggers an

immune rejection process. In the case of bone marrow transplantation,

the new immune system will be formed by transplanted cells of the

donor. Therefore, the rejection will be donor versus receptor (graft

versus host disease, GVHD). Allotransplants and xenotransplants are

maintained by immunosuppressive therapies, which are aggressive and

have to be maintained for long periods. Notwithstanding this drawback,

allogeneic transplants are necessary to correct pathologies associated

with modiﬁcations in the genome, mutations leading to cancer, or in

which the patient’s cells are not able to promote repair and tissue

regeneration.

Heterotopic autologous therapies are at present the most frequently

explored in basic, preclinical, and clinical research. Since 1990, the multi-

potency of adult stem cells, mainly from the bone marrow, has allowed

the possibility of repair and regeneration of different tissues using the cells

of the patient. The best example of heterotopic autologous therapy is the

use of stem cells from the bone marrow in the treatment of cardiac

ischemia. The rationale for this application is the capacity of such stem

cells to differentiate in situ into cell lineages of the receptor site. The main

goal of this kind of therapy is to offer tissue repair as close as possible to

the physiologically normal tissue without restrictions of histocompatibil-

ity, which is a problem in allotransplants and xenotransplants. It is hoped

that in the future these therapies will be able to restore an entire organ,

such as pancreas, heart, or kidney, without the immune rejection and the

required immunosuppressive treatments.

20.2 Primary material

The concept of cellular therapy involves manipulation and use of cells as a

primary material, and this requires extensive knowledge of their structure

and function (Orkin, 2000). A cell from any part of an organism does not

exist as an isolated entity. It coexists in a three-dimensional system, highly

organized and physiologically dependent upon the reciprocity of cell–cell

and extracellular matrix–cell interactions. The understanding of all the

elements related to the organization of a tissue structure is the major

challenge for biological engineering. The tissue structures must be recon-

structed, or at least equivalent structures have to be generated, to grant

tissue function, using essentially three elements: the cells, the molecular

mediators that promote and control cell growth, differentiation and tissue

integration, as well as the three-dimensional matrices or scaffolds that

provide the spatial organization.

476 Animal Cell Technology

20.2.1 Stem and mature cells

Any stem cell is deﬁned by being able to maintain its own population by

self-renewal and to differentiate into one or several types of mature fully

differentiated cells. Stem cell proliferation can be modulated between low

and high proliferation kinetics. Embryonic stem cells have a high prolif-

eration rate, since they must rapidly construct an entire new organism.

Adult stem cells are generally quiescent or proliferate slowly, but they can

be mobilized upon demand, and induced to actively proliferate, as occurs

with hematopoietic stem cells (Kondo et al., 2003). The properties of stem

cells are associated with their ability to undergo symmetrical or asymme-

trical division (Figure 20.1). Classical symmetrical divisions are operational

in the self-renewal or expansion of the original stem cell pool. Asymme-

trical division depends upon the existence of intracellular segregated

microdomains and organization of genetic elements, as observed in ovo-

cytes. This organization allows micro-compartmentalized elements to be

partially or totally segregated during cytoplasm division upon mitosis.

Thus, a stem cell (mother cell) in mitotic division can generate an identical

unit and another one with a different proﬁle (daughter cell), which is

generally engaged and committed into differentiation along a required

pathway.

Differentiation

Self-renewal

Endosteal stromal cell

Subendosteal stromal cell

Asymmetric division

Figure 20.1

Schematic representation of an asymmetric cell division. This ﬁgure shows a

hematopoietic stem cell under the inﬂuence of cell components within its niche.

Between the stromal endosteal and subendosteal cell layers, the hematopoietic

stem cell is stimulated into distinct polarities that split cytoplasmic components

into microdomains. The cell division that is able to promote a different distribution

of these intracellular elements, generates distinct offspring. The asymmetric

division explains the phenomena of simultaneous self-renewal and differentiation.

Cell therapies and stem cells 477

Stem cells can be characterized into three main classes: embryonic,

germinal, and somatic or adult stem cells. Embryonic stem cells (ESCs) are

derived from early stages of an embryo, from the morula, or from the

inner cell mass of the blastocyst. Both are able to produce an entire

organism. From the morula, the source of cells can be derived during

blastic cleavages, which occur just after fertilization. Each cell up to the

stage of 2 cells in humans, and up to 32 cells in animals, depending upon

the species, is able to generate a complete new embryo. On the other hand,

stem cells can be harbored by isolating the inner cell mass of blastocysts.

This stage corresponds to the period of implantation into the uterus. In

humans, they may be obtained from left-over embryos generated by in

vitro fertilization.

The cells of the inner cell mass can take part in the generation of all the

tissues with the exception of the extra-embryonic structures such as

trophoblast and placenta. They are the main target of the worldwide

research on ESCs. To obtain these cells requires disintegration of the

blastocyst. This fact has generated ethical and religious questions concern-

ing human embryos.

Moreover, scientists are still concerned about how to control the high

capacity of ESCs for proliferation and their apparently erratic differentia-

tion. The goal is to be able to control maturation towards a predetermined

cell lineage. These issues have to be solved before the use of ESCs in

humans, particularly because of their high tendency to develop into

teratocarcinomas.

The germinal stem cells or EGCs (embryonic germ cells) are found in

reproductive tissues during embryo development. They populate gonads

that harbor progenitors of gametes. Similar cells are found in male adult

gonads. Apparently, they have the potential to generate multiple cell

lineages, but the disadvantage for therapy is the difﬁculty of obtaining

these cells, which would involve invasive surgery during embryo develop-

ment. This is associated with further ethical questions.

Cells derived from an embryo implanted into a recipient are recog-

nized as non-self, and upon transplantation they are identiﬁed by the

recipient’s immune system. Thus, transplantation causes rejection, and

this limits their use in cell therapy. Advances in reproductive techniques

have emerged recently with the possibility of therapeutic cloning as a

new option for cell therapy. The proposal is to substitute the nucleus of

an ovocyte or an embryonic cell established in vitro, with the nucleus of

a somatic cell from the recipient, generating new ESC lineages. The

ovocyte or embryonic progenitor cells will be able to combine the

capacity to generate any tissue within the host genome, with the appro-

priate immunologic characteristics to prevent rejection. However, the

defects of the genome will also be transferred, thus preventing the use of

this technique for genomic defects. Nevertheless, the extensively manipu-

lated nuclei generally develop unpredictable random chromosomal de-

fects, which may modify the cellular phenotypes. However, in view of

the size of the genome and the interactivity of its diverse regions, an

assessment of the potential dangers of any alteration is not feasible, and

the medical risk of using progenitor cells generated by cloning in human

478 Animal Cell Technology

beings is unacceptably high. Furthermore, human cloning is technically

difﬁcult and also involves many religious and ethical dilemmas. There-

fore, somatic or adult stem cells seem to be the best current solution for

cellular therapies.

Somatic (from the Greek somatikos ¼ bodily) stem cells are present

in almost all the tissues (Jiang et al., 2002a). The most well-known

system is the hematopoietic tissue. The hematopoietic system in adults

is lodged within the bone marrow and its stem cells are speciﬁcally

located in the endosteal region, that is, close to the internal bone

surface. Moreover, bone marrow has at least three stem cell lineages:

hematopoietic, mesenchymal, and endothelial stem cells (Figure 20.2).

Among these, mesenchymal stem cells display the broadest capacity for

differentiation. Mesenchymal cells are associated with the inner cell

mass and are not in direct contact with the external environment.

Although these cells are most frequently of mesodermal origin, they can

also be derived from the ectoderm and endoderm, in a process termed

the ‘‘epithelio-mesenchymal transition.’’ Mesenchymal stem cells are

able to restore tissue structural elements, called the cellular stroma and

the corresponding extracellular matrix. They inﬂuence differentiation of

other stem cells, as described below. Descriptions of many mesenchymal

cell populations in different tissues can be found in the scientiﬁc

literature, but there are controversies as to whether they belong to

different lineages (Figure 20.2). The fact that mesenchymal cell progeni-

tors can circulate in blood makes this question even more difﬁcult.

Mesenchymal stem cells are apparently pluripotent and, therefore, they

can generate many cell types, with the possible exception of germline

cells (Jiang et al., 2002b).

Studies of stem cell progression towards the completely differentiated

mature cells have already identiﬁed several intermediary precursors, orga-

nized in a cascade (Shizuru et al., 2005). The best known and studied

cellular differentiation cascade is the hematopoietic system (Figure 20.3).

Within hematopoiesis, it is possible to identify many intermediary pre-

cursors between the hematopoietic stem cell and mature blood cells. This

identiﬁcation is based mainly on the phenotypic proﬁle of cellular surface

proteins, using ﬂow cytometry as the main tool. This is a relatively simple

technique that involves coupling a monoclonal antibody (mAb) with a

ﬂuorescent marker (ﬂuorochrome). In this way, diverse cellular markers

can be combined and thus a cellular subpopulation can be deﬁned, as

shown in Figure 20.3.

The ideal protocol for tissue regeneration is the use of autologous cells

in the heterotopic context. This approach allows the stem cells to produce

a tissue or organ for autologous repair and tissue regeneration. The ﬁrst

step is to deﬁne the primary source and to identify the cells to be

transplanted. Later, it is necessary to ensure that the transplanted cells

recognize the target tissue and acquire the speciﬁc functions. This is the

object of cell therapy in vivo, or bioengineering ex vivo or in vitro.Itis

necessary to understand the physiology of the cells to be generated. That

is: how, where, when, and what it is necessary to do to convert the stem

cells into functional differentiated tissue cells.

Cell therapies and stem cells 479

Epiblast

cell

Ectodermic

progenitor

Endodermic

progenitor

Mesodermal

progenitor

Mesenchymal

precursor

PDMPC

MAPC

MSC

Pericyte

Endothelial

Lymphatic

progenitor

precursor

Meso-angioblast

Hemangioblast

Hematopoietic

progenitor

(LT-HSC)

mice

Lin

KDR

Brachyury

CD41

CD105

-/lo

mice

Lin

KDR

CD117

CD34

-/lo

Brachyury

mice

Lin

KDR

CD117

CD106

CD31

-/lo

Tie-2

mice

Lin

KDR

CD117

CD144

CD31

Sca-1

vWF

-/lo

Tie-2

CD146

CD34

KDR

CD117

CD44

CD90

Sca-1

SSEA-I

CD13

Tie-2

CD45

CD13

CD44

CD90

CD73

CD105

HLA-II

Lin

MHC-II

Lin

CD34

CD31

CD45

HLA-DR

mice

Lin

KDR

CD144

CD31

vWF

-/lo

Tie-2

CD146

hum

Lin

KDR

CD133

CD117

CD34

CD144

CD31

CXCR4

vWF

-/lo

CD146

hum

Lin

KDR

CD133

CD34

CD144

CD31

CXCR4

vWF

-/lo

CD146

Osteoblast

Chondroblast

Tendocyte

Adipocyte

Fibroblast

Skeletal

muscle

Cardiac

muscle

Oligodendrocyte

CBF 1 Opn Ocn ALP

CBF 1 ColI/IX

FGF-Rs BMP-12R Col.II

-SMA Tropomyosin

CD100 CD81 GM-1

kx-2.5 cTn-I c-Tn-T -MHC

PPAR- LPL

Col I FGF-Rs ?

Smooth

muscle

Reticul ocyte

(myofibrobl ast)

-SMA SM22 Desmin -MHC

= Totipotent !?

mice

hum

Stro-1

CD49

CD54

CD105

CD56

CD106

CD90

CD73

CD146

Stro-1

CD13

HLA-I

CD29

CD105

CD33

CD166

Lin

CD34

CD104

CD45

HLA-DR

CD133

CD62L

Lin

CD34

CD14

CD45

bone marrow

umbilical cord

Endothelial

precursor

Endothelial

Cell

++++

+++

480 Animal Cell Technology

Figure 20.2

Schematic representation of the mesenchymal and endothelial cascade of differentia tion in a murine model (mice) and in humans (h um). All

mesenchymal and e ndothelial cells are ne gative for the antigen CD45. Based on this, the dynamics of surface antigen expression along

development of different mature cells d erived from the mesenchymal and endothelial systems can be observe d. Rounded arrows indicate self-

renewal potential. Smooth, thinner arrows indicate directions of cellular differentiation, and dotted arrows indicate possible hierarchies, but yet

to be proved experimentally. The question marks indicate lack of data on the pathway or on cellular identity. The identiﬁcatio n of CDs (clusters

of differentiation) and other antigen cell markers can b e found in the list of abbreviations: LT-HSC, long- term hematopoietic stem cell; EC,

endothelial cell; PDMPC, placental-derived mesenchymal progenitor cell; MSC, mesenchymal stem cell; MAPC, mesenchymal adult progenitor

cell.

Cell therapies and stem cells 481

LT-HSC ST-HSC MPP

CMP

CLP

mice

Lin

CD117

CD90

Sca-1

CD135

CD133

VEGF-R1

CD31

-/lo

mice

Lin

CD117

Sca-1

CD90

CD127

CD34

CD16/32

-/lo

mice

Lin

CD117

Sca-1

CD90

CD127

CD34

CD16/32

-/l o

mice

EPO-R

CD127

CD34

CD16/32

GM-CSF-R

+/hi

mice + hum

EPO-R

CD127

CD34

CD16/32

CD110

+/hi

hum

Lin

CD133

CD7

CD45RA

hum

Lin

CD133

CD33

CD13

hum

CD123

CD117

CD34

CD13

CD33

mice

Lin

CD117

Sca-1

CD90

CD135

CD133

-/l o

mice

Lin

CD117

CD90

Sca-1

CD135

-/l o

hum

Lin

CD117

CD90

CD133

-/lo

GMP

MEP

Pro-T

Pro-NK

T lymphocyte

NK cell

B lymphocyte

Dendritic

lymphoid

Dendritic

myeloid

Astrocyte

Osteoclast

Macrophage

Neutrophil

Eosinophil

Bas ophil

Mast cell

Platelets

Erythrocyte

Pro-B

Pro-DC

Monocyte

Polymorphonuclear

Megakaryocyte

Reticulocyte

(erythrocyte)

mice

hum

CD44 CD25 CD117

CD34 CD1a CD38 CD117

+- +

+-- -

mice

hum

CD3 CD16 NK1.1 DX-5

CD3 CD16 CD56

-+ ++

-++

mice

hum

CD11c CD11b CD8

+-+

CD11c HLA-DR CD8

mice

hum

CD11c CD11b CD8

++-

CD11c HLA-DR CD8

mice

hum

CD11b CD60 GFAP

++ +

CD14 CD60 GFAP

mice

hum

CD11b Gr-1 CD16/32 F4/80

+lo/+ + +

CD11b CD14 CD64

mice

hum

CD11b Gr-1 IgE-R 7/4

++ ++

CD66a CD35 CD87 IgE-R

++ +

mice

hum

IL-5R Gr-1 IgE-R MMP-9

++ + +

CD14 CD125 IgE-R

+++

mice

hum

CD11b Gr-1 IgE-R MMCP-8

++ + +

CD11b CD14 IgE-R CD203c

+++ +

mice

hum

CD11b Gr-1 IgE-R MMCP-4

++ + +

CD34 CD14 IgE-RI CD123

++ + +

mice

hum

Te r - 1 1 9

CD235a CD71

+-/lo

mice

hum

CD135 MHC-II CD11c

CD4 HLA-DR CD7 CD3

mice +hum CD3 CD4/8 TCR

mice +hum CD19 BCR

mice + hum CD11b CD11c RANK

+-+

mice+humCD41

mice

hum

CD110 EPO-R

CD110 EPO-R CD71

mice

hum

CD110 EPO-R

CD110 CD34 CD38 CD41

++++

mice

hum

CD43 B220

CD34 CD10 CD19 TdT

++--

mice

hum

CD122 NK1.1 DX-5

cCD3 CD7

+++

G-CSF-R

M-CSF-R

-lo

482 Animal Cell Technology

Figure 20.3

Schematic representation of the hematopoietic cascade in mi ce and human (hum). All the cells of the hematopoietic system are positive for

antigen CD45. Based on this, the dynamics of surface antigen expression along development of di fferent mature cells derived from the

hematopoietic stem cell system can be observed. Rounded arrows indicate self-renewal potential. Smooth thinner arrows indicate directions of

cellular differentiation. The identiﬁcation of CD s (clusters deﬁnition) and other antigen cell markers are listed in the glossary. (LT-HSC - long

term hematopoietic stem cell; ST-HSC - short term hematopoieti c stem cell; MPP - multipotent progenitor; CLP - common lymphoid

precursor; CMP - common myeloid precursor; GMP - granu locyte-mo nocyte p recursor; MEP - megacaryocyte-erythrocyte precursor; T - T

lymphocyte; B - B lymphocyte; NK - natural killer cell; DC - dendritic cell).

Cell therapies and s tem cells 483